The present invention relates to the field of plant molecular biology, more particularly to the regulation of gene expression in plants.
Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Thus, where expression is desired in a preferred tissue of a plant, tissue-preferred promoters are utilized. In contrast, where gene expression throughout the cells of a plant is desired, constitutive promoters are the regulatory element of choice. Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in expression constructs of transformation vectors to bring about varying levels of tissue-preferred or constitutive expression of heterologous nucleotide sequences in a transgenic plant.
Frequently it is desirable to have preferential expression of a DNA sequence in a tissue of an organism. For example, increased resistance of a plant to insect attack might be accomplished by genetic manipulation of the plant""s genome to comprise a tissue-specific promoter operably linked to a heterologous insecticide gene such that the insect-deterring substances are specifically expressed in the susceptible plant tissues. Preferential expression of the heterologous nucleotide sequence in the appropriate tissue reduces the drain on the plant""s resources that occurs when a constitutive promoter initiates transcription of a heterologous nucleotide sequence throughout the cells of the plant.
Alternatively, it might be desirable to inhibit expression of a native DNA sequence within a plant""s tissues to achieve a desired phenotype. In this case, such inhibition might be accomplished with transformation of the plant to comprise a tissue-specific promoter operably linked to an antisense nucleotide sequence, such that tissue-specific expression of the antisense sequence produces an RNA transcript that interferes with translation of the mRNA of the native DNA sequence in a subset of the plant""s cells.
Phloem tissue transports nutrients, hormones, and other substances throughout the various plant organs. The parenchyma cells of phloem participate in loading and unloading sucrose and other nutrient content into the phloem transport system. The high nutrient content of sap located in phloem tissue causes the phloem to be the target of a variety of insect species, including aphids (family Aphididae), corn borers (family Pyralidae), and leafhoppers (Cicadellidae) among others. Damage to plants resulting from infestation by these insects occurs through multiple mechanisms, including loss of nutrients and water to the insects, introduction of virus particles into the phloem tissue following infestations, and creation of tissue susceptible to fungal attack. A need exists for vascular tissue-preferred promoters operably linked to heterologous nucleotide sequences that help protect a plant against pathogens such as insects, viruses, fungi, nematodes, and the like.
An additional need exists for a vascular tissue-preferred promoter sequence would be operably linked to a heterologous nucleotide sequence that modifies the loading characteristics of vascular tissue and thereby affects plant development and maturation, carbon allocation, and crop yield. By altering the levels of substances involved in phloem loading, the loading characteristics of vascular tissue, plant development and plant growth can be influenced. A need exists for promoter sequences that can be used to modulate expression of substances that regulate vascular tissue loading.
There may also be a use for a vascular tissue-preferred promoter in improvement of stalk strength. For example, via cell wall thickening such as by deposition of more cellulose. A vascular tissue-preferred promoter is desirable to use for expressing genes that are involved in cellulose biosynthesis in order to increase cell wall strength. When cell wall strength is increased in corn stalk, better standability is expected. This is particularly relevant to improving resistance to stalk lodging in corn. An example of such application is to use a vascular tissue-specific promoter to drive a cellulose synthase gene that is involved in secondary cell wall formation, such as the irx3 gene from Arabidopsis thaliana (Taylor N G, Scheible W R, Cutler S, Somerville C R, Turner S R. 1999. The irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis. Plant Cell 11:769-80). In this case, cells that do not normally have secondary wall would potentially gain additional cell wall growth, thus leading to a stronger cell structure.
Thus, isolation and characterization of phloem-preferred promoters that can serve as regulatory regions for tissue-preferred expression of heterologous nucleotide sequences of interest are needed for genetic manipulation of plants to exhibit specific phenotypic traits.
Compositions and methods for regulating expression of heterologous nucleotide sequences in a plant are provided. Compositions comprise novel promoter sequences that initiate transcription in a vascular tissue-preferred manner, particularly a phloem-tissue preferred manner. Specifically a transcriptional initiation region isolated from a Prunus serotina gene encoding prunasin hydrolase is provided. Further compositions of the invention comprise the nucleotide sequence set forth in SEQ ID NO.: 1 and a fragment of the nucleotide sequence set forth in SEQ ID NO.: 1. The compositions of the invention further comprise nucleotide sequences having at least 70% identity to the sequence set forth in SEQ ID NO.: 1 or a fragment thereof, and nucleotide sequences that hybridize under stringent conditions to any one of the above-mentioned sequences. The sequence set forth in SEQ ID NO.: 2 represents a modification of the nucleotide sequence made for cloning purposes. The sequence for the prunasin hydrolase operon including the prunasin hydrolase promoter region is set forth in SEQ ID NO.: 3. Nucleotides 989-2626 of SEQ ID NO.: 3 encode a prunasin hydrolase polypeptide. SEQ ID NO.: 4 is the amino acid sequence for the prunasin hydrolase polypeptide. SEQ ID NOS: 5 and 6 are related variations of the polynucleotide sequence disclosed as SEQ ID NO.: 1.
Compositions of the present invention also include a DNA construct comprising a promoter sequence of the invention operably linked to a nucleotide sequence of interest, wherein the promoter is capable of driving expression of the nucleotide sequence in a plant cell. Transformed plant cells, transformed plants, and transformed seeds comprising the novel promoter sequences of the invention are also provided.
Methods for expressing a nucleotide sequence of interest in a plant are provided. The methods comprise stably incorporating into the genome of a plant cell an expression cassette comprising a promoter sequence of the invention operably linked to a nucleotide sequence of interest, wherein the promoter is capable of initiating transcription of the nucleotide sequence in a plant cell. The methods further provide a means for preferentially expressing a nucleotide sequence in vascular tissue, more particularly phloem tissue.